JPS5815924A - Immunological adsorbent and adsorbing apparatus - Google Patents

Abstract

PURPOSE:An adsorbent, used in a container having an inlet and outlet for a fluid, and prepared by joining a hydrophobic compound to a carrier consisting of a crosslinked polymer containing vinyl alcoholic units as constituents, and capable of specifically adsorbing and removing globulin compounds. CONSTITUTION:An adsorbent in which a hydrophobic compound having a solubility <=100 millimoles/dl (25 deg.C) in physiological saline solution or a polymer containing the hydrophobic compound or both are joined to a carrier consisting of a crosslinked polymer, e.g. a crosslinked polyvinyl alcohol obtained by hydrolyzing a copolymer consisting of a vinyl ester of a carboxylic acid with a vinyl compound having an isocyanurate ring, having vinyl alcoholic units as main constituents is packed in and held by a gap between filters 3 and 3' in an adsorbing apparatus 1 for globulin compound having a fluid inlet 5 and fluid outlet 7 to form an adsorbent layer 9, which is used to adsorb immunoglobulin and/or immunoglobulin complex specifically.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a globulin-based compound that specifically adsorbs and removes immunoglobulins and/or immunoglobulin complexes that are thought to be closely related to various diseases caused by the immune system of the body. The present invention relates to an adsorbent and a device for adsorbing and removing globulin compounds using the adsorbent.

As is well known, immunoglobulins and/or immunoglobulin complexes expressed in the blood are associated with cancer, immunoproliferative syndromes, rheumatoid arthritis, autoimmune diseases such as systemic lupus erythematosus, allergies, rejection reactions during organ transplants, etc. It is thought that there is a close relationship with the cause or progression of diseases and phenomena related to the body's immune function.

Therefore, by specifically adsorbing and removing the above-mentioned immunoglobulins and/or immunoglobulin complexes from body fluid components such as blood and plasma, it is possible to prevent the progression of the above-mentioned diseases, alleviate symptoms, and hasten healing. That was expected.

As a result of intensive research in accordance with the above request, the present inventors have surprisingly found that a hydrophobic compound and/or an oligomer containing a hydrophobic compound bound to an insoluble carrier adsorbs immunoglobulin with extremely high activity. In particular, we discovered that it specifically adsorbs autoantibodies and immune complexes.
A patent application was previously filed (%Application No. 7152, 1983).

The present invention was made as a result of a more detailed study on carriers in relation to the previous invention, and relates to improvements in carriers.

Hitherto, no carrier specifically designed for this purpose is known. Therefore, there was no choice but to repurpose a carrier that is generally known for use in affinity chromatography. Known carriers include natural polymer carriers such as agarose carriers, dextran carriers, and cellulose carriers, polyacrylamide carriers, and glass carriers.

However, when using natural polymer carriers for therapeutic purposes,
It has the following drawbacks.

There are many operational restrictions due to insufficient tl+ mechanical strength. For example, the carrier may be destroyed during preparation of the adsorbent such as activation or immobilization, or the carrier may break or crumble during transportation or use.

(2; Because it is a soft gel, it is packed into a column and used for extracorporeal circulation.
When K is used, the body fluid containing the substance to be removed cannot be passed through at a high flow rate. When a high viscosity, high solute concentration liquid such as body fluid is flowed at a high flow rate, since it is a soft gel, the filling volume decreases, causing clogging and a decrease in flow rate, which may eventually stop flowing.

(3) Since it is a soft gel and does not have permanent pores, it cannot be easily sterilized, which is an essential requirement for an adsorbent for extracorporeal circulation therapy. For example, in the case of chemical sterilization such as ethylene/oxide gas sterilization, the material is sterilized by freeze-drying, but the pores are destroyed by freeze-drying and do not return to their original state even if they are re-dispersed in an aqueous medium.

During freeze-drying, its volume decreases to about half, and even when it is redispersed in an aqueous medium, it only returns to about 8.0% of its original volume, and its adsorption capacity usually decreases. Some methods include adding additives to protect the pores during freeze-drying, but to prevent the additives from entering body fluids, they must be thoroughly washed before use. Furthermore, heat sterilization such as high-pressure steam sterilization cannot be used because it destroys the pores. Similarly, radiation sterilization cannot be used as it destroys the skeleton and pores.

(4) Furthermore, when natural polymeric carriers are used for extracorporeal circulation therapy, they are said to activate the complement system and the coagulation system, resulting in leucovenia, slo/bocytovenia, etc., and are therefore not very desirable.

- Although polyacrylamide-based carriers have the advantage of being relatively physically and chemically stable, nonspecific adsorption of plasma proteins occurs and residual toxicity of acrylamide cannot be ignored. Although glass-based carriers are physically and chemically stable, they cause significant non-specific adsorption of plasma proteins and cause thrombus formation when used for whole blood, so they cannot be used. It is commonly used in the problem of a carrier based on conventional technology, and is generally popular, and adsorbs imnoglobulin and / L or imnoglobulin complexes in high activity and specific, maintains stable activity. It is an object of the present invention to provide an adsorbent material that is flexible, easy to sterilize, and suitable for body fluid purification or regeneration, and an adsorption device using the same.

The present inventors have carried out research in accordance with the above objectives, and have bonded hydrophobic compounds and/or polymers containing hydrophobic compounds to carriers of various foods, and have determined the binding activity for immunoglobulins and immunoglobulin complexes and the plasma protein As a result of evaluating the non-specific adsorption and usability for whole blood, it was surprisingly discovered that a copolymer containing vinyl alcohol units as a main component gave extremely good results as a carrier. It was completed.

That is, the present invention provides an immunoglobulin characterized in that a hydrophobic compound and/or a polymer containing a hydrophobic compound is bonded to a carrier made of a crosslinked copolymer mainly composed of vinyl alcohol units. 9. An adsorbent for immunoglobulin and/or immunoglobulin complexes, and an adsorbent for immunoglobulin and/or immunoglobulin complexes, characterized in that the adsorbent is accommodated in a container having a fluid inlet and outlet. It concerns an adsorption device.

The carrier used for this purpose is required to have interaction characteristics with plasma proteins and adsorption characteristics, such as (1) low non-specific adsorption of plasma proteins, and (2) no activation of the complement system or coagulation system. In addition, in terms of safety, it is required that 1) it is sterilizable, 2) it has physical strength and does not cause chipping or scum of the carrier, and 2) there is no eluate from the carrier. Furthermore, when used as an adsorbent for whole blood, interaction with blood cell components, ie, thrombus formation and non-specific adhesive residual blood of blood cell components, etc., becomes a problem. Natural polymer carriers containing sugars such as dextran, agarose, and cellulose interact with blood cells and plasma components, activating the complement system and coagulation system. On the other hand, synthetic polymer carriers are said to be relatively free from these problems. The present inventors investigated synthetic polymer carriers and found that a carrier made of a copolymer containing vinyl alcohol units as a main component successfully solves the above-mentioned unresolved problems.

Due to its hydrophilic nature, the carrier made of a crosslinked copolymer mainly composed of vinyl alcohol units has a small interaction with solutes such as proteins in plasma, and reduces nonspecific adsorption to a minimum. It also has extremely excellent properties such as not interacting with the complement system and coagulation system in plasma. In terms of physical properties, it has heat resistance, enables heat sterilization, and has excellent physical and mechanical strength, which is a characteristic of synthetic polymers.

When used as a carrier for adsorbent for whole blood, it has extremely excellent properties such as having little interaction with blood cell components, minimizing thrombus formation, non-specific adhesion of blood cell components, and residual blood. .

The higher the density of hydroxyl groups in the crosslinked copolymer of the present invention, the more its hydrophilicity increases, which is advantageous in minimizing interaction with blood components, and also increases the hydroxyl group density when activated with an activation reagent. It is preferable because the active group density can be maintained at a high level, but
On the other hand, physical and mechanical strength decreases in relation to crosslinking density (crosslinking agent content). Therefore, the □hydroxyl group density is preferably 5 to 17 meq.l, more preferably 6
It's been 15 days.

The hydroxyl group density can be determined by reacting the carrier with acetic anhydride in a pyridine solvent and measuring the amount of acetic anhydride consumed by the reaction with the hydroxyl group or the change in weight of the carrier. When 1 g of the dry carrier is reacted with 1 wwnoL of acetic anhydride, the hydroxyl group density is 1 wwnoL.

A crosslinked polymer mainly composed of vinyl alcohol units can be synthesized by polymerizing a monomer having a hydroxyl group or introducing a hydroxyl group through a chemical reaction of the polymer. It is also possible to synthesize both in combination.

As the polymerization method, a radical polymerization method can be used. The crosslinking agent may be introduced by copolymerization during polymerization, or by chemical reaction between polymers (between polymers, □ polymer and crosslinking agent), or both may be used in combination.

For example, it can be made by copolymerizing a vinyl monomer and a vinyl or allyl crosslinking agent. Examples of the vinyl monomer in this case include vinyl esters of carboxylic acids such as vinyl acetate and vinyl propionate, and vinyl ethers such as methyl vinyl ether and ethyl vinyl ether.

Examples of crosslinking agents include allyl compounds such as triallyl isocyanurate and triallyl cyanurate, di(meth)acrylates such as ethylene glycol dimethacrylate and diethylene glycol dimethacrylate, butanediol divinyl ether, diethylene glycol divinyl ether, and tetravinyl. Polyvinyl ethers such as glyoxal, polyallyl ethers such as diarylidene pentaerythritol and tetraallyloxyethane, and glycidyl acrylates such as glycidyl methacrylate can be used. If necessary, a copolymer of other comonomers can also be used.In the case of a vinyl copolymer, a vinyl compound (allyl compound) having a vinyl ester of carboxylic acid and an incyanurate ring is copolymerized. A crosslinked product of triallyl isocyanurate of polyvinyl alcohol obtained by polymerization and hydrolytic decomposition of a copolymer provides a particularly good carrier in terms of strength and chemical stability.

Although the case of vinyl copolymer was exemplified above, the present invention company,
It is not limited to this.

The shape of this carrier is spherical, granular, filamentous, hollow fiber,
Although any shape such as a flat membrane can be effectively used, a spherical shape or a granular shape is particularly preferably used in view of the surface area of the carrier (adsorption capacity as an adsorbent) and the flow of body fluid during extracorporeal circulation. - Therefore, the known suspension polymerization method is particularly effective (used) as a method for synthesizing the carrier.

The average particle size of the spherical or particulate carrier is 25 to 2500 μm
However, depending on its specific surface area (adsorption capacity as an adsorbent) and body fluid circulation surface, it should be 150 to 1500 μm.
Particularly preferred are those.

In the present invention, a crosslinked copolymer having a specific surface area of at least 5 d/9 is used.

Specific surface area is expressed as the surface area occupied by nitrogen gas adsorbed per unit weight of dry crosslinked copolymer. In other words, the specific surface area indicates how effectively the substance constituting the crosslinked copolymer per unit weight forms the surface in a dry state.

Generally, a crosslinked copolymer swells in a medium that is compatible with the crosslinked copolymer and shrinks when dried.

In the case of a soft gel in which the medium-filled bores are maintained only by cross-linked networks during swelling, the networks collapse when the gel dries, and the bores almost disappear. In this case, the specific surface area is the value only for the outside of the particle, so the agarose conventionally known for affinity chromatography, which generally has a low value of less than 1 dy', is a soft gel. Therefore, the bore disappears due to drying.

Therefore, it is not easy to sterilize, and furthermore, it has a soft mesh that is easily crushed, so even when it is packed into a column and used for extracorporeal circulation, body fluids cannot be passed through it at a high flow rate for a long period of time.

On the other hand, in the case of a hard gel (crosslinked copolymer) that has a rigid structure and can withstand freeze-drying and heat sterilization, the bore will shrink a little when it dries, but the swelling state will remain almost unchanged. maintain. In other words, it has permanent pores, and its specific surface area is higher than that of soft gels.1, at least 5 w? It showed a value of 79 or more.

The specific surface area of the present invention was determined by the most common BET method using nitrogen gas. In addition, the sample used for specific surface area measurement must be sufficiently dried, but the crosslinked copolymer of the present invention may be difficult to dry.
After the water-wetted carrier was equilibrated with acetone, it was dried under reduced pressure at 60° C. or lower and subjected to measurement.

It is appropriate that the water retention capacity (hereinafter referred to as wR) of the crosslinked copolymer used in the present invention is in the range of 0.5 to 169/9,
Preferably 1.0 to 15.09. /9 range.

wR is when the crosslinked copolymer is equilibrated with physiological saline,
WRu is the amount of physiological saline that can be contained in the particles expressed as the value of the dry weight of the crosslinked copolymer.
This is a guideline for the weight within the crosslinked copolymer. As WR increases, the weight of the part that forms the skeleton per unit volume of the crosslinked copolymer in water, that is, the weight of the crosslinked copolymer itself, decreases in an increasing manner, and therefore, in physiological saline and even body fluids,
The mechanical strength of the crosslinked copolymer decreases. Furthermore, as WR decreases, the weight per unit weight (or unit volume) effective for adsorption decreases, resulting in a decrease in adsorption capacity. Therefore, it is preferable for the carrier of this purpose that wR is in an appropriate range.
After measuring, the crosslinked copolymer that has been sufficiently equilibrated with physiological saline is centrifuged to remove the physiological saline adhering to the surface of the crosslinked copolymer, and its weight (wl) is measured. It can be determined by the formula.

The exclusion limit molecular weight (protein) of the carrier is 150,000 to 10,000,000 since the molecular weight of the target adsorbent of the present invention is 150,000 (IgG) and reaches 10,000,000 for immune complexes, especially IgM immune complexes. preferable. The most general exclusion limit for purposes of the present invention is 14-1 to 5 million asci.

The adsorbed substances targeted by the present invention are immunoglobulins and/or immunoglobulin complexes, and to explain in more detail, ordinary immunoglobulins, rheumatoid factors, anti-nuclear antibodies, anti-DNA antibodies, anti-9'7 protein Immunoglobulins and reduced products of immunoglobulins, including autoantibodies such as erythrocyte antibodies, anti-erythrocyte antibodies, anti-platelet antibodies, acetylcholine receptor antibodies, serum desorption antibodies, anti-spinal globulin antibodies, pile microseam antibodies, and anti-colon antibodies. , immunoglobulin derivatives such as chemically modified products, complexes between immunoglobulins or immunoglobulins and other substances, especially antigens and antigen-like substances (hereinafter collectively referred to as globulin compounds). 11 Preferred adsorbent substances targeted by the invention are autoantibodies and immune complexes that are closely related to the cause and progression of autoimmune diseases.

The hydrophobic compound 1 used in the present invention refers to a compound having a solubility in physiological saline of 100 mmol/dt or less (at 25°C), more preferably 30 mmol/d or less. A compound having a solubility in physiological saline of more than 100 mmol/di becomes too hydrophilic and has a reduced affinity for globulin compounds, resulting in an extremely reduced adsorption capacity. Furthermore, an affinity for albumin, which is more hydrophilic, is generated, and albumin is also non-specifically adsorbed, which is undesirable.

Among the hydrophobic compounds, compounds having at least one aromatic ring give particularly favorable results.

Aromatic ring means a cyclic compound with aromatic properties,
All can be usefully used, including benzene-based aromatic rings such as benzene, naphthalene, and phenanthrene; nitrogen-containing six-membered rings such as pyridine, quinoline, acridine, inquinoline, and 7-enantridy; indole, carbazole, isoindole, and indolizine. , phorufilin, 2,3.2',
1. Nitrogen-containing 5-membered ring such as 3'-pyrrolopyrrole. Polyvalent nitrogen-containing 6-membered rings such as pyridazine, pyrimidine, 5yrn-)riazine, sym-tetrazine, quinazoline, 1,5-naphthyridine, pteridine, phenazine, pyrazole, imidazole, 1,2,4-triazole, L2,3- Polyvalent nitrogen-containing 5-membered rings such as triazole, tetrazole, benzothiazole, imidazole, and purine, norharman ring, perimidine ring, oxygen-containing aromatic rings such as benzofuran, isobencyphine, and dibendofuran, pendothiophene,
Sulfur-containing aromatic rings such as chenothiophene and chepin, oxygen-containing heteroaromatic rings such as oxazole, isoxazole, 1.2.5-oxadiazole, pe/zooxazole, thiazole, inthiazole, 1.3.4 - A hydrophobic low-molecular organic compound having at least one aromatic ring such as a sulfur-containing heteroaromatic ring such as thiadiazole or benzothiazole or a derivative thereof gives preferable results.

Among them, compounds containing an indole ring such as tryptamine,
Gives particularly favorable results. This can be interpreted as a result of the hydrophobicity, steric structure, and molecular rigidity of the compound acting effectively in the bonding between the globulin compound and the compound.

In addition, as a result of intensive research in search of inexpensive hydrophobic compounds that can be used more safely and practically, the present inventors have found that hydrophobic amino acids and their derivatives have been found to be highly effective and specific in globulin production. We have discovered that it can adsorb and remove system compounds.

Hydrophobic amino acids and their derivatives are defined by Tanford
.

Nozaki (J, Am, Chem, Soc, ,
184 4240 (1962), J. Biol, C.
hem, 246 2211 (1971)) (Journal of American Chemical Science, 1st edition, 4240 (1962), Journal of Biological Chemistry 246
．． Amino acids and their derivatives with a hydrophobicity scale of 1500 cal/no1 or more as defined by 221H1971)) with a solubility in physiological saline of 100 mmol/l
It means a compound with d1 or less. Examples include lysine, valine, leucine, tyrosine, phenylalanine, inleucine, tryptophan and derivatives thereof. Among these hydrophobic amino acids and their derivatives, tryptophan and its derivatives give particularly good results. Furthermore, amino acids can be used without particularly limiting the conformation of tSd.

In addition, as a result of intensive research, the present inventors have surprisingly found that among hydrophobic compounds, non-dissociable, amphoteric, and cationic compounds when bound to the carrier, We have discovered that globulin compounds can be adsorbed highly selectively. That is, the anionic compound had an affinity for albumin, contrary to the expected result since albumin molecules are more electronegative than globulin molecules, and the amount of adsorption of globulin compounds decreased. For example, phenylalanine methyl ester, phenylalanine, and tyrosine do not differ greatly in their hydrophobicity, but when their amine groups are covalently bonded to a carrier and their immunoproplyne adsorption ability is evaluated, the ranking of phenylalanine methyl ester, phenylalanine, and tyrosine is An example of this can be seen in the decreased adsorption capacity of phenylalanine and tyrosine. In addition, non-aromatic ring compounds also showed similar results.One compound line in particular has an anionic group and a C1 sulfonic acid group, making it more hydrophilic. This is presumed to be due to the interaction between the charge of the compound, albumin, and the charge of the microenvironment within the molecule.

In view of the above experimental results, the mechanism of action of the present invention is as follows:
This is thought to be based on the hydrophobic interaction force (Van der Waals force) between the hydrophobic compound bound to the carrier and the globulin compound. In addition, it is presumed that charge amount interaction force (Coulomb force) acts secondarily as a delivery force.

The polymer containing the hydrophobic compound of the present invention is a polymer having a molecular weight of 10,000 or less, preferably a molecular weight of 1000 or less. This results in protein A (molecular weight 42,000
) handling during immobilization compared to natural polymers such as
It can also be easily stored after fixation. Furthermore, even if the skeletal substance is eluted from the carrier, a polymer with a molecular weight of 10,000 or less has negligible antigenicity to living organisms, is safe, and can be easily sterilized.

The polymer can be obtained by copolymerizing the hydrophobic compound monomer alone or with other compounds. As the hydrophobic compound, for example, a vinyl derivative of a compound containing an indole ring such as tryptamine, or a hydrophobic amino acid such as tryptamine can be used.

Methods for bonding a hydrophobic compound or/and a polymer containing a hydrophobic compound to a carrier made of a crosslinked copolymer mainly composed of vinyl alcohol units include covalent bonding, ionic bonding, physical adsorption, embedding, or polymerization. Any known method such as precipitation and insolubilization on the combined surface can be used, but in view of the elution properties of the bound substance, it is preferable to use it after fixation and insolubilization by covalent bonding. Therefore, known methods for activating immobilized enzymes and carriers commonly used in acquisition chromatography can be used.

Examples of activation methods include cyanogen halide method, epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformer method, 1,1'-carbonyldiimidazole method, etc. I can do it. The activation method of the present invention is limited to the above examples as long as it can perform a substitution and/or addition reaction with a 21-nucleophilic reactive group having active hydrogen such as an amino group, a hydroxyl group, a carboxyl group, or a thiol group of the bond. It's not a thing.

If necessary, a molecule (spacer) of any length may be introduced between the crosslinked copolymer (carrier) and the hydrophobic compound or/and the polymer containing the hydrophobic compound. For example, by reacting and bonding the hydroxyl group of the carrier with the isocyanate group on one side of hexamethylene diinocyanate, and then reacting and bonding the remaining incyanate group with the amine group, hydroxyl group, thiol group, carboxyl group, etc. of the bond. It can be implemented. As for the spacer length, a range from no spacer to 20 atoms in the spacer gives particularly preferable results.

In the present invention, if necessary, two or more hydrophobic compounds or/and a polymer containing a hydrophobic compound may be bound to the carrier.

In the present invention, the amount of the hydrophobic compound or/and the polymer containing the hydrophobic compound bound to the carrier is 0.000.
A range of 1 to 30 mg is preferred. 22- Preferably in the range of 0.5-15. Retention amount is 0.
If it is less than 1.2 mm, the adsorption amount of globulin compounds will be extremely reduced, and if it is more than 30.0 mm, the adsorption specificity will be decreased, which is not preferable.

The globulin-based compound adsorption device of the present invention is made by filling and holding the globulin-based compound adsorbent as described above in a container provided with an inlet/outlet for body fluids.

In FIG. 1, reference numeral 1 shows an example of the globulin compound adsorption device of the present invention, in which a body fluid inlet 5 is connected to an opening at one end of a cylinder 2 through a backing 4 with a filter 3 stretched inside. A cap 6 having a body fluid is screwed into the opening at the other end of the cylinder 2, and a cap 8 having a body fluid lead-out lower part is screwed into the other end opening of the cylinder 2 through a backing 4' having a filter 3' stretched inside to form a container. The adsorbent layer 9 is formed by filling and holding an adsorbent in the gaps 3 and 3'.

The adsorbent layer 9 may be filled with the adsorbent of the present invention alone, or may be mixed or laminated with other adsorbents. As other adsorbents, for example, adsorbents for other malignant substances (antigens) such as DNA, activated carbon having a wide range of adsorption capacities, etc. can be used. As a result, a wider range of clinical effects can be expected due to the synergistic effect of the adsorbent. The volume of the adsorbent layer 9 is 50 to 400++t/! when used for extracorporeal circulation. The degree is appropriate.

When the device of the present invention is used for extracorporeal circulation, there are two methods as follows: One method is to separate blood taken from the body into plasma components and blood cell components using a centrifuge or membrane plasma separator.The plasma components are passed through the device, purified, and then separated into blood cells. One method is to return the blood to the body together with its components, and the other method is to pass blood taken from the body directly through a nuclear device and purify it.

In addition, regarding the passage speed of blood or plasma, since the adsorption efficiency of the adsorbent is extremely high, the particle size of the adsorbent can be made coarser, and the degree of packing can be lowered, so the shape of the adsorbent layer can be changed. Regardless, high passing speeds can be provided. Therefore, a large amount of body fluid can be treated.

The method for passing body fluids may be either continuous or intermittent, depending on clinical needs or equipment conditions.

As described above, the adsorbent and adsorption device of the present invention adsorb and remove globulin compounds in body fluids at a high rate and specifically, and are extremely simple, simple, and safe.

Because a crosslinked copolymer whose main component is vinyl alcohol units is used as a carrier, it has extremely excellent properties such as low nonspecific adsorption of plasma tannin and low interaction with the complement system and coagulation system. , has excellent physical and mechanical strength, and has extremely low chipping and scum due to adsorbent preparation and handling.Also, because it is hard, body fluids can flow at high flow rates.Furthermore, it has heat resistance, so it Sterilization methods (ethylene oxide gas sterilization, heat sterilization such as high-pressure steam sterilization, gamma ray sterilization, etc.) can be carried out easily and reliably.Furthermore, when used as an adsorbent for whole blood, blood cell components can be easily and reliably carried out. This has the advantage of minimizing thrombus formation, non-specific adhesion of blood cell components, residual blood, etc. because the interaction with the blood is small.

The present invention is applicable to the general use of purifying and regenerating body fluids such as autologous plasma, and is suitable for safe and reliable treatment of diseases related to the body's immune function, especially autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. In addition to being effective in treating diseases, the adsorbent of the present invention can be used as a therapeutic device by filling it into a device, and can also be used to separate immunoglobulins, autoantibodies, immunoglobulin derivatives, and immune complexes. It can also be used extremely effectively as an adsorbent for purification and as a substrate for these measurements.

Embodiments of the present invention will be explained in more detail with reference to Examples below.

26- Example 1 Vinyl acetate 100f,) realyl isocyanurate) 2
4j t (X=0.20), ethyl acetate 1241,
Heptane 124f, polyvinyl acetate (degree of polymerization 500)
A homogeneous mixture of jl f and 2,2'-azobisisobutyronitrile 6.11, polyvinyl alcohol - weight %, sodium dihydrogen phosphate dihydrate 0.05
Weight % and disodium hydrogen phosphate dodecahydrate 1.5
400 kg of water dissolved in 100 ml of water were placed in a flask, stirred thoroughly, and heated and stirred at 6.5°C for 18 hours and then at 75°C for 5 hours to carry out suspension polymerization to obtain a granular copolymer. Ta.

After filtration, washing with water, and extraction with acetone, caustic soda 46
．． 5? and methanol 2, 40
The transesterification reaction of the copolymer was carried out at ℃ for 18 hours.

The average particle size of the obtained particles was 150 μm. When the hydroxyl group density (qOH) was determined using the above method, 1Srne
It was q/f. ~ This gel was packed into a stainless steel column with an inner diameter of 7,511 m and a length of 25 α, and aqueous solutions of dextran and polyethylene glycol with various molecular weights, albumin,
When a phosphate buffered salt solution of immunoglobulin G1, immunoglobulin M, and β-lipoprotein was measured, each was eluted in descending order of molecular weight. The exclusion limit molecular weight of dextran was about 3 x 1 O5, and the exclusion limit molecular weight of protein was about 18 x 106. Also 0.3M
When a solution of human r-globulin and human albumin was passed through an aqueous solution containing sodium chloride and 0.1M sodium phosphate as a solvent, the amount was recovered at a recovery rate of almost 100, indicating that the gel was not adsorbed nonspecifically. were very few. All sample measurements were performed at a flow rate of 1 gIt/jlll
Gel 5 was then transesterified and thoroughly washed with water.
Suspend 0 cc in 200-ml water, add St cyanogen bromide, and stir. p using a 2N aqueous sodium hydroxide solution.
The reaction was carried out for 8 minutes while keeping H at 10 to 11. After the reaction was completed, it was immediately filtered through a glass filter and then washed with 2 portions of water to obtain an activated gel. A hydrophobic compound was bound to the activated gel by a conventional method, and excess active groups were blocked with ethanolamine. The retained amount was determined by converting the primary amine group of the remaining hydrophobic compound into 4-phenylspiro[furan-2(5H), 1'-phthalane]-5. Reactively bonded with S'-dione ("Fluram ■" manufactured by Rossi), 4
It was calculated by measuring with fluorescence of 75 to 490 nm (excitation wave 590 nm) and subtracting the amount of physical adsorption separately tested with unactivated Sepharose 4B.

Adsorbent after ethanolamine blocking, pH 4-
After repeated washing with 0.1M acetate buffer and pH 8.5-0.1M boric acid buffer, the plate was washed with physiological saline, drained, and used for experiments.

In the adsorption experiment, 6 volumes of human plasma and 1 volume of adsorbent were mixed, and 67
It was incubated at ℃ for 3 hours. Globulin after adsorption,
A/G test killer 1- (A/G
B test Measured by Wako, Wako Pure Chemical Industries (copper).In addition, an adsorption experiment was conducted using unactivated gel and used as a control.The results are the adsorption rate when the control adsorption rate is set to 0. It is shown in Table 1. From Table 1, it is clear that the hydrophobic compound bonded to the crosslinked copolymer mainly composed of 4 units of vinyl alcohol-29 adsorbs globulin specifically and at a high rate. It is.

Example 2 An experiment similar to Example 1 was conducted except that 1-tryptophan methyl ester was used as the hydrophobic compound and human rheumatoid arthritis patient plasma was used instead of human plasma. Rheumatoid factor (autoantibody), a malignant substance in rheumatoid arthritis
, the adsorption-30-removal ability of the immune complex was measured. Rheumatoid factor was measured using a latex agglutination test and a passive sensitization hemagglutination test.

The latex agglutination test is a detection method that measures the property of latex particles agglutinating when patient plasma containing rheumatoid factor is applied to polystyrene latex particles adsorbed with human γ-globulin. A dilution series is created and the rheumatoid factor concentration is evaluated at the plasma dilution rate at which latex particles no longer aggregate.

Plasma containing a high concentration of rheumatoid factor does not have a high dilution rate to become negative, whereas plasma with a low concentration becomes negative.

Passive sensitization hemagglutination test
- Globulin is adsorbed, but the rest is the same as the latex agglutination test. Generally, the passive sensitization hemagglutination test is considered to have higher specificity than the latex agglutination test.

- A dilution series was prepared with glycine saline buffer, and a latex agglutination test was negative for rheumatoid factor using a kit from the Japan Lyophilization Research Institute. Similarly, evaluation was performed using the CRAHA test (passive sensitization hemagglutination test) and Fuji Organ Pharmaceutical Co., Ltd. (made of copper).

In addition, measurement of immune complexes can be performed using polyethylene glycol,
By complement hemolysis. In this method, immune complexes precipitated with polyethylene glycol are reacted with complement in the serum of a healthy human, and the amount of remaining complement is measured by the amount of hemolysed red blood cells bound to antibodies. It evaluates body mass.

The operating method and conditions for this method are as follows.

Pour il + sample 0.3- into a separate tube. 0.2M ED
Add 50μ of TA and stir. Oxalic acid buffer (PBS
) Add 50μ and stir. 12.51 Add 0.1% of PEG (polyethylene glycol) (Mw 750G), stir, and place at 4°C for 90 m Apparatus 2) Centrifuge at 4°C for 100 m, and centrifuge the resulting precipitate at 2.5 ml of PE01- Wash with 0-. 17θθf15-centrifuge and drain supernatant.

+3137°C GVB+ (gelatin veronal buffer containing divalent cations) 50a13 is added to dissolve the precipitate. Pooled healthy human serum 101Llj is added as a source of complement. The immune complex and complement are reacted at 37° C. and 301 m.

1411.5 X 10'/m EA (antibody-sensitized red blood cells)1. Add Od and shake at 37°C for 60 minutes to promote hemolytic reaction due to residual complement.

(5) After the reaction, add 4°C saline 6,5- and centrifuge.
Measure the absorbance (OD414) of the supernatant.

(6) Calculate the inhibition rate of hemolysis relative to the control (serum from a healthy person), and set the unit to PEG-cc.

[Inhibition] Control-Specimen Absorbance Stop A=-, In addition, globulin in Example 1
It was measured by the method used in . An unactivated gel was used as a control, and the results are shown in Table 2-35 in terms of the removal rate when the adsorption rate of the control was set to 0. From this, the /, -) liptophan bound to the carrier of the present invention. It is clear that methyl esters efficiently adsorb globulin compounds in plasma, especially rheumatoid factors and immune complexes. In addition, the complement values before and after adsorption were measured, and in both cases the decrease was small.

Example 5 The adsorbent 5- of Example 2 was stored in a container as shown in FIG.
Created a globulin removal device.

A globulin adsorption experiment was conducted using the experimental system shown in FIG.

That is, healthy human blood (heparin added 1
2DOU/100-blood) 11 is pumped out at a flow rate of 1-per-minute by a pump 12, passed through a drip camber 15 and a sampling port 13, and transferred to a container 10. The tube 14 was arranged so that it could be returned to. Note that 250 U of heparin was added to the blood.

After blood was circulated for 1 hour using the above device, the blood was sampled and the amount of globulin in the plasma was measured. The results are shown in Table-3.

In all cases, there was a relatively small decrease in white blood cells and platelets before and after circulation. There was still relatively little thrombus formation and residual blood.

Table-3

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional view showing one example of the globulin compound adsorption apparatus of the present invention, and FIG. 2 is an explanatory diagram of a model experiment in an example. 1... Globulin compound removal device 2...
...Cylinder 3.3'...Filter 4.4
′・・・Backing 5・・・Body fluid inlet
6... Cap 7... Body fluid outlet
8... Cap 9... Adsorbent agent Takeshi Shimizu Figure 1 Figure 2 Figure 4

Claims (1)

[Claims] 1. A hydrophobic compound and /l or a polymer containing a hydrophobic compound are bonded to a carrier made of a crosslinked copolymer mainly composed of vinyl alcohol units. An adsorbent for immunoglobulins and/or immunoglobulin complexes. 2. The crosslinked copolymer mainly composed of vinyl alcohol units is a crosslinked polyvinyl alcohol obtained by hydrolyzing a copolymer of a vinyl ester of carboxylic acid and a vinyl compound having an isocyanurate ring. The adsorbent according to scope 1. 3. A container having an inlet and outlet for fluid containing an adsorbent in which a hydrophobic compound and/or a polymer containing a hydrophobic compound is bonded to a carrier made of a crosslinked copolymer mainly composed of vinyl delcol units. 1. An adsorption device for immunoglobulins and/or immunoglobulin complexes, characterized in that the adsorption device is housed in an immunoglobulin and/or an immunoglobulin complex.